|Ph.D Student||Tal Nir|
|Subject||Insights into the Molecular Mechanism of BtuCD-F,|
The Vitamin B12 Transporter of Escherichia coli
|Department||Department of Medicine||Supervisor||Professor Oded Lewinson|
|Full Thesis text|
The E. coli transporter BtuCD-F uses ATP to translocate vitamin B12 across the inner membrane, from the periplasm to the cytoplasm. The complete transporter consists of three parts: BtuF, the vitamin B12 binding protein; BtuC2, a membrane-embedded permease; and BtuD2, an ATPase. The latter has two ATP-binding cassettes (ABCs) that jointly compose two ATPase sites. The ATPase, BtuD2, together with the permease, BtuC2, form an ABC transporter. ABC transporters are ubiquitous proteins that translocate a wide range of substrates. Importantly, they are implicated in human diseases. Here we studied BtuCD-F, to gain insights into its workings in particular, and those of other ABC transporters in general. Since the ATPase part is common to all ABC transporters, we focused on ATP hydrolysis by BtuD2, and how it relates to vitamin B12 transport. We found that the two ATPase sites of BtuCD cooperated in ATP hydrolysis; that they were dependent upon one another; but also, that one ATPase site was adequate for vitamin B12 utilization by E. coli. Positive cooperativity and interdependence in ATP hydrolysis has been demonstrated before, thus suggesting that the molecular mechanism of the ATPase is similar amongst ABC transporters. Hence, any differences in function would be the result of the coupling between the ATPase and the permease, the permease itself, or other factors in the overall mechanism of the transporter (such as the substrate binding protein, regulatory domains, and else). Also, we studied BtuCD-F complex formation in spheroplasts, a more natural context than in vitro. We found that BtuCD bound BtuF with sub-nM affinity and that BtuCD-F complex formation was decreased in the presence of vitamin B12, thus corroborating the previous in vitro results.